Spring11-MCRO255 Lab_1 Lecture - Welcome to MCRO255 Lab...

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Unformatted text preview: Welcome to MCRO255 Lab MCRO255 Your TA’s Your Myra dela Pena [email protected] Megan Wicks Megan [email protected] Martha Clark [email protected] Richard Watkins [email protected] Megan Richard
7 10 9 8 6 Front of classroom Where you sit today is your Where assigned seat for the rest of the semester! the Door
1 2 3 4 5 Myra Martha When you enter the room… When
• Book bags, coats, etc. in front of the room • Wash your hands with soap and water, followed Wash by an alcohol-based hand sanitizer by • Use Bac-down and paper towels to wipe down Use bench top before placing materials down bench • We will start lab on time, so arrive early!
– If you know you will be late regularly please talk to If your TA your What you will need What
• • • • • • • • Close-toed shoes Lab coat Safety glasses Gloves Plastic bag to store lab coat, goggles, etc. Lab Manual Pen or pencil Sharpie (for labeling) • No food, gum, or drinks permitted in lab! Safety Precautions Safety
• Please see your TA before the 2nd week of class if you have any of the following conditions: have – Pregnancy – Immune suppression from chemotherapy, Immune splenectomy, sickle-cell disease, HIV, etc. – Allergic hypersensitivity to penicillin, sulfa drugs, Allergic latex, etc. latex, • ABSOLUTELY NO EATING or DRINKING in the lab ABSOLUTELY room. • In this class you will be working with live human In pathogens. With proper techniques, the risks are minimal. minimal. Attendance Attendance
• If you know in advance that you have to miss a lab contact If BOTH your lab instructor and Bruce Alexander ( BOTH [email protected]) You may arrange with your TA to attend You either the Wednesday or Thursday lab. either • If you are sick please do not come to lab. However, all excused If absences require a doctor’s note and notification by e-mail to require notification your respective TA by the end of that day. by • 1 unexcused absence or 2 tardy arrivals will result in the unexcused subtraction of 1 point from your participation grade. subtraction • You will still be expected to turn in your pre-lab for the week You that you missed. that Cheating Cheating
• Cheating is unethical and WILL NOT be tolerated in this Cheating class. class. • If there is any suggestion that you have engaged in or If attempted to engage in cheating you will be approached by one of us and Dr. Cramer will be notified. We are required to file a report with the UNC honor system if a violation is suspected. • Cheating includes plagiarism. It is illegal to copy a Cheating sentence or a significant portion of a sentence from a written source (such as your lab manuals) and use it in your own writing (such as pre-labs). Lab Grading Lab
• • • • • 3 quizzes (20+20+25) Required Unknowns 11 Pre-lab assignments Attendance and participation Total 65 points 20 points 11 points 4 points 100 points *** There are 3 possible bonus points. Pre-lab Write-ups Pre-lab Plagiarism will not be tolerated Lab Quizzes Lab
• Questions will be drawn from what we cover in lab. • The quiz will start at the beginning of class. The The classroom door will be closed when we start the quiz. If you show up late, you will have to knock to be admitted, and you will not receive any additional time to complete the quiz. the • Put your name, drawer number, and your TA’s name on Put your quiz. your • The 3rd and final quiz will be CUMULATIVE Lab Quizzes Lab
• If you miss a quiz, you will be allowed to make it up if If you have an excused absence. You need to email us on Thursday or Friday to schedule your make-up quiz, and you must make up the quiz by Tuesday of the following week. • You will NOT be allowed to make up a quiz for an You unexcused absence. unexcused Labeling Labeling
• With almost 200 students in this class, it is VERY, VERY With important to label your plate correctly important • Labeling code: Labeling Ex: T-4A MC E. coli 1-13-10 Ex:
Day Day Seat Initials Pathogen Date • We will instruct you where to place plates at end of each We lab lab Lab 1: Microscopy and the Observation of Stained Specimens Materials for Today’s Lab Materials
1. Slide box containing slides of Escherichia coli and Staphylococcus aureaus 2. Immersion oil & lens paper 3. Light microscope Light Microscope: Magnification Magnification
• Magnification: size of the object – Total magnification= (magnification of objective lens) x (magnification of ocular lens) – Ocular lens= 10X – 4 objective lenses= 4X, 10X, 40X, 100X (oil) 4X x Objective lens 10X Ocular lens = 400X Total magnification Resolution Resolution
• Resolution: The ability to distinguish between two points that are close together. that Image of pollen grain with good resolution Image (left) and poor resolution (right) (left) http://science.howstuffworks.com/light-microscope.htm/printable Magnification + Resolution
100X 100X Magnification alone Magnification with Magnification resolution resolution When to use immersion oil? When
• Required to get good resolution for magnification Required above 60X above • Use ONLY with 100X objective lens • Apply oil to the top of cover slip on the slide Why use immersion oil?
Refractive index: bending of light as it changes Refractive from one medium to another (air to water) from -Refractive index of air is less -Refractive than glass so light rays bend light -Immersion oil has a similar -Immersion refractive index as glass so it decreases bending of light decreases -This increases resolution of -This the image the Outline of Today’s lab Outline
1. Identify the parts of a microscope 1. View 2 prepared slides of bacterial specimens using oil View immersion immersion 2. View 4 examples of bacterial stains at the View demonstration stations demonstration 3. View a live preparation of 2 bacteria using a phase View contrast microscope contrast 1. Identify the parts of a microscope Identify
• Pages 8-9 in lab manual • See TA with any questions Today’s lab Today’s
1. Identify the parts of a microscope 1. View 2 prepared slides of bacterial specimens View using oil immersion using 1. View 4 examples of bacterial stains at the View demonstration stations demonstration 2. View a live preparation of 2 bacteria using a phase View contrast microscope contrast 2. View 2 prepared slides of bacterial specimens using oil immersion specimens
At your desk there is a small box with 2 slides of bacterial At specimens (E. coli and S. aureus) that have been specimens E. S. that prepared for you by heat-fixing and gram-staining prepared The Gram Stain The
• Most commonly used stain for bacteria Most • Gram-negative = red red Gram-positive = purple purple Follow the instructions in your lab manual (p. 10,11) manual
• Adjust fine focus to the center of its range of motion • Use ONLY the fine focus adjustment with the 10X, 40X Use and 100X objectives and • Use only a small amount of oil for the 100X objective! Follow the instructions in your lab manual (p. 10,11) manual
• Have your TA check to make sure that you have Have correctly located the bacterium. correctly • Use the lens paper (not Kim-Wipes!!!) to wipe off the oil Use lens from the slide and lens. from • Place slide back in box. Do not throw away! Place Do Today’s lab Today’s
1. Identify the parts of a microscope 2. View 2 prepared slides of bacterial specimens using oil View immersion immersion 1. View 4 examples of bacterial stains at the View demonstration stations demonstration 1. View a live preparation of 2 bacteria using a phase View contrast microscope contrast 3. View 4 examples of bacterial stains
• There are 4 microscopes at each demonstration There table that have bacterial specimens stained using 4 different techniques: using • Negative Stain • Acid Fast Stain • Spore Stain • Capsule Stain Negative Staining Negative
• Bacteria are negatively Bacteria charged, therefore when cells are stained with negatively charged acid stains such as nigrosin the background rather than the cells are stained cells • Air dry instead of heat fix • Used to identify Used Cryptococcus sp. in CSF of patients with meningitis of
Cryptococcus neoformans. India ink staining of CSF. (600X) Acid Fast Staining Acid
• For bacteria that can’t be stained with Gram stain, such as For Mycobacteria Mycobacteria • For these type of bacteria cells are not decolorized easily For with acid-alcohol with • Method 1. Primary stain (pink) 2. Decolorizing agent (acid-alcohol) 3. Secondary/ counter stain (blue) Spore Staining Spore
• Some bacteria form a metabolically inactive spore as a Some long-term response to stress long-term
– – – Clostridium tetani : tetanus Clostridium tetanus Clostridium botulinum: botulism Clostridium botulism Bacillus anthracis: anthrax Bacillus anthrax Example: Bacillus subtilis Example: Bacillus
1. Primary stain (malachite green)Primary endospore endospore 1. Water wash removes stain from Water vegetative cell vegetative 1. Counterstain (safranin) stains Counterstain vegetative cell vegetative Capsule Staining Capsule
• Capsule – Located outside cell wall – protective against immune protective cell recognition (anticell phagocytic) • Stain generates pink Stain bacterium surrounded by light blue “halo”, which is the capsule capsule – Fuchsin primary stain – Alcian blue counterstain Klebsiella pneumoniae Today’s lab Today’s
1. Identify the parts of a microscope 2. View 2 prepared slides of bacterial specimens using oil View immersion immersion 3. View 4 examples of bacterial stains at the View demonstration stations demonstration 1. View a live preparation of 2 bacteria using a phase View contrast microscope contrast 4. View live bacteria using phase contrast microscopy contrast
• Visualize unstained bacteria (live bacteria) • Visible contrast created as light passes through Visible various cellular components (different refractive indexes) indexes) Today in lab: living preparation of mixed Today bacteria, E. coli and S. aureus S.
E. coli- flagella result in E. directional motility of the bacteria bacteria S. aureus- move by Brownian S. motion which is characterized by seemingly random movement seemingly Before you leave the room today… today…
• Place dirty lens paper and paper towels in trash cans Place with orange autoclave bag orange • Wipe down lab bench with Bac-down and paper towels • Dispose of gloves in orange autoclave bag Dispose orange • Wash your hands with soap and water, followed by an Wash alcohol sanitizer alcohol Good websites for microscopy theory theory
• http://science.howstuffworks.com/light-microscope.htm • http://www.microscopy-uk.org.uk/mag/indexmag. html? http://www.microscopy-uk.org.uk/mag/artjul03/ http://www.microscopy-uk.org.uk/mag/artjul03/ gocompmic.html gocompmic.html • http://web.uvic.ca/ail/techniques/scope_basics.html ...
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